There was a time in graduate school when Peter Novick wasn’t sure if his research would lead anywhere. As a graduate student in cell biologist Randy Schekman’s laboratory at the University of California at Berkeley, Novick had been using a genetic approach to understand the yeast secretory pathway, responsible for moving proteins out of the cell. Now a professor at the University of California, San Diego, Novick says it was only when he identified a mutant in which vesicles piled up inside the cell, showing that it had a defective secretory pathway, that he knew he had made a breakthrough. “That convinced me. Before then I wasn’t sure if I had a thesis project, afterwards it was pretty clear I did,” he says.

It turned out that Novick had a lot more than a thesis project. His graduate work paved the way for Schekman’s detailed analysis of the secretory pathway, work for which Schekman shared the 2013 Nobel Prize in Physiology or Medicine. “Peter Novick was instrumental in starting our genetic approach to the secretory pathway in yeast,” says Schekman. “Already as a first year graduate student, he showed great skill and devised the screen and a selection procedure that allowed us to isolate the first mutant, sec1-1, and within another year, mutations representing 23 genes that defined the major stages in the secretory pathway,” he says.

In eukaryotic cells, such as yeast and higher organisms, the secretory pathway transports proteins from the endoplasmic reticulum (ER), an organelle where the proteins are synthesized, to the Golgi apparatus, a cellular compartment that serves as a sorting station. From the Golgi, the proteins then move to the cells’ plasma membrane. The latter step involves transporting the proteins in vesicles that fuse with the plasma membrane and release proteins to the outside of the cell. The regulation of such intracellular trafficking is relevant to many human diseases, including cancer and diabetes. Understanding how yeast secretes proteins has also been used in biotechnology to produce proteins such as insulin.

Novick says his graduate work led to his continued interest in teasing apart the many components of the secretory pathway. Using a combination of yeast genetics and cell biology, he has spent his career investigating the tightly regulated mechanisms involved in intracellular transport. For his contributions to our understanding of this fundamental physiological process, Novick was elected to the National Academy of Sciences in 2013.

“In his own lab, first at Yale and now at UC San Diego, Peter launched a brilliant independent career with the discovery that a protein called Sec4 encodes a small GTP-binding protein, the first of three dozen so-called Rab proteins that we now know control the targeting of transport vesicles to all the many destinations in the cell,” says Schekman. “On the strength of this work and much more in subsequent years, he was elected to the National Academy of Sciences, an honor that was in my opinion long overdue,” he says.

Identifying Genes Involved in Secretion

When he went to graduate school at Berkeley, Novick was interested in studying cell membranes and membrane proteins, but “there weren’t too many labs studying membranes at that time,” he says. Then he met Randy Schekman. “He was a brand new assistant professor and he suggested the possibility of using genetics to study membranes,” Novick says. Novick enjoyed genetics, so “the idea of doing genetics on membranes was very appealing,” he says, and “It turned out to be a really good match.”

In Schekman’s laboratory, Novick set out to make yeast mutants in which secretion was blocked. When he selected for mutants that had a defective secretion system, “I got this excellent sec1-1 mutant, and I spent quite a bit of time studying it,” Novick says (1). Blocking secretion is often lethal to cells, so all of the mutants Novick got were temperature sensitive, including sec1-1.

“At 25 degrees [Celsius] it was essentially like wild-type cells, and then within minutes of shifting to 37 degrees [Celsius], secretion was blocked,” he says. “It just was like turning off a light switch,” Novick says. “The mutant cell became packed with vesicles that couldn’t get out. In terms of a graphic, dramatic experiment, that takes the cake,” he says. “I don’t think I ever got a better mutant than sec1-1, it was just a beautiful mutant,” Novick says.

Novick studied the sec1-1 mutant in detail and found an unusual property: “These guys were far, far more dense than any wild-type cells,” he says. “It makes sense. If you’re not expanding the surface area of the plasma membrane, the volume of the cell can’t increase, but the cells were still metabolically active and making RNA and protein, so they got very dense,” Novick says.

Novick was able to use the fact that the mutants with defective secretion were dense to enrich them 100-fold over WT cells. Over a couple of different experiments, he isolated more than 200 secretory mutants, which later defined 23 genes involved in the secretory pathway (2).

Novick was confident that these secretory pathway genes represented a conserved eukaryotic mechanism. “We didn’t know for sure how general these pathways would be, but there’s general optimism that things evolve once, and if they work, you keep them,” he says. This view was reinforced when he found that the secretory pathway he described was strikingly similar to one previously described in pancreatic cells (3, 4).

“In my thesis I put these genes on a map, and that map looked strikingly like the secretory pathway in pancreatic acinar cells. They both involved transport from the ER to Golgi to secretory vesicles to the cell surface, with energy requirements at each of those stages,” Novick says. “Considering how different yeast is from the pancreas, I just thought that was great,” he says.

However, finding the genes involved in the secretory pathway was only a starting point. “There was tremendous promise in that we had identified all these genes, but when I left graduate school we still had no idea what any of the gene products were doing,” he says. “It was a black box, with a lot of gene products, but we didn’t know what they were,” Novick says.

Hunt for Gene Function

In 1985, Novick started his own laboratory at Yale University and decided to use the rapidly evolving field of molecular genetics to investigate what the different genes in the secretory pathway were doing. “Yeast transformation had been developed, and it became clear that we could get at the genes without having to purify proteins,” he says (5).

Novick decided to focus on the end of the secretory pathway, involving the transport of vesicles from the Golgi to destinations such as the plasma membrane. Ten of the 23 mutants he had identified as a graduate student had defects in the end of the secretory pathway, and Novick used molecular genetics to analyze the genes involved. One gene product he identified, Sec4, turned out to be particularly interesting.

Sec4 was similar to the Ras family of GTPases, which play a key role in many cell signaling events, and it turned out to be one of the first members of what is now called the Rab family of GTPases (6). Rab proteins have been found to control and coordinate the various biochemical reactions involved in membrane traffic. Novick’s model was that Sec4 acted as a kind of a master regulator of intracellular trafficking. “That turned out to be remarkably on target,” he says.

As Novick systematically identified all of the other Sec genes that functioned at the end of the secretory pathway, he noticed that many of the gene products seemed large and peripherally associated with membranes, leading him to hypothesize that the proteins might associate with one another.

Novick’s laboratory went on to show that 6 of the 10 Sec genes that were found to act at the end of the secretory pathway encoded proteins that were all part of the same complex, which could associate with membranes. “This ultimately became the complex that we call the exocyst,” Novick says. Two additional components, Exo70 and Exo84, were identified biochemically.

Novick showed that the exocyst complex acted as a downstream effector of Sec4 to direct the fusion of secretory vesicles to the plasma membrane and was one of the first Rab effectors found. Novick also found that one of the other Sec proteins, Sec2, interacted with Sec4 and catalyzed Sec4’s exchange of GDP for GTP, a form of cellular energy currency similar to ATP. Sec2 was one of the first Rab exchange proteins identified. Sec4 thus acted as the Rab GTPase controlling the exocyst, and Sec2 as the exchange protein that activated Sec4.

Coordinating the Secretory Pathway

Novick’s next goal was to determine how the various components of the secretory pathway could be coordinated. His laboratory identified an interaction between Sec2 and a Rab GTPase called Ypt32 that acted just upstream of Sec4. Sec2 was found to bind to the activated form of Ypt32 as an effector (7). As Sec2 also bound to Sec4 as an exchange protein, this led to the idea of a “cascade model,” where one Rab binds to the exchange protein that activates the next Rab. “This gave us a really attractive model for how you direct transport along the pathway, where each Rab recruits its own set of effectors, as well as the exchange protein that activates the next Rab,” Novick says. Once the next Rab is activated, it recruits a different set of effectors, which then activate the next Rabs in a programmed series.

Novick also showed that there was a counter-current cascade. “Not only is a Rab recruiting the protein that activates the next Rab, it’s also recruiting the protein that inactivates the preceding Rab,” Novick says (8). “That’s formed an attractive model that has gotten a lot of attention lately, and quite a few proteins are showing up that seem to fit that paradigm,” he says.

Novick’s Inaugural Article further elucidates the regulation of Sec2 and the final stages of the secretory pathway (9). Novick found that phosphorylation of a binding site on Sec2 switches Sec2 from binding Ypt32 to binding one of the subunits of the exocyst, Sec15. When Sec2 is bound to Ypt32, it is part of the cascade of Rab activation. However, when Sec2 switches to binding Sec15, it can start a positive feedback loop where Sec2 activates Sec4, Sec4 recruits Sec15, Sec15 is still bound to Sec2, and Sec2 can now reactivate Sec4.

“Our thought is that Sec2 is initially recruited in a nonphosphorylated state by Ypt32, and as the vesicle is reaching the plasma membrane, phosphorylation acts as a regulatory switch that causes Sec2 to now bind Sec15 and start this feedback loop that’s required to prepare the vesicle for fusion with the plasma membrane,” he says. “It’s all part of the circuitry, and this is a switch that switches from one type of circuit to a different type,” Novick says.

Novick states that researchers’ understanding of vesicle trafficking has come a long way since he was in graduate school. “Randy Schekman winning the Nobel Prize together with James Rothman and Thomas Sudhof really highlights that we know most of the players in the secretory system, and we know mostly how they act in terms of one specific event, such as fusion of a vesicle,” he says.

“What’s really challenging now is understanding how you coordinate all of these different events to give rise to a functional pathway,” Novick says. The cascades that Novick and others are currently studying represent one way to coordinate events and tie them together into a concerted functional pathway. However, there is still a lot left to learn, Novick says. “Understanding how all of these events are really coordinated in a cell, to give rise to a functioning pathway, that’s the challenge for the field.”

Footnotes

This is a Profile of a recently elected member of the National Academy of Sciences to accompany the member’s Inaugural Article on page 19995 in issue 50 of volume 110.

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